MicroRNA (miRNA) has emerged as a post-transcriptional regulator of gene expression impacting - among other factors - cell growth and differentiation, and the progression of multiple genetic diseases. miRNA processing in the cell involves two ribonucleoprotein complexes composed of RNase III enzymes and double-stranded RNA binding domain (dsRBD) chaperones that work together to achieve two highly specific endonuclease cleavages. The central hypothesis of this proposal is that the specificity inherent to the miRNA processing activity of these multi-protein complexes is a direct result of dsRBD interactions: the specific recognition of the miRNA precursors by the dsRBDs in the complexes and the unique juxtaposition of the multiple dsRBDs. There are at least ten dsRBDs involved in miRNA production: two in DGCR8, one in Drosha, one in Dicer, three in TRBP, and three in PACT. Bioinformatics approaches have vastly increased our knowledge of miRNA prevalence and function, and cellular and molecular biology approaches have provided a general, but powerful, overview of miRNA biochemistry and function. Structural biology techniques have been extensively applied to the proteins involved in processing small interfering RNA (siRNA) in unicellular organisms, and the results have been extrapolated to partially explain miRNA processing in multicellular organisms. However, only the second endonuclease-mediated maturation step necessary for production of functional miRNA is shared with the simpler siRNA pathway, where the mode of function is also mechanistically distinct. Thus, much remains to be worked out for miRNA maturation. This project will take a holistic structural biology based approach, aiming to establish the molecular mechanism of miRNA processing by both the Microprocessor complex - unique to miRNA processing - and the complexes shared between the miRNA and siRNA pathways. The first aim of the project is to provide atomic resolution structures for each of the dsRBDs involved in miRNA processing, which have not previously had their structures determined, to define the conformational dynamics of each by NMR spectroscopy, and to quantify the intrinsic RNA binding affinity of each dsRBD in isolation. In the second aim, the role of the dsRBDs from the RNase III endonucleases Drosha and Dicer in providing specificity to the cleavage reactions they catalyze will be explored through binding assays and NMR spectroscopy. Three of the five proteins to be studied contain more than one dsRBD - as do both of the ribonucleoprotein complexes composed by them - and so aim 3 will seek to define the role of cooperative interactions among the dsRBDs in yielding affinity and specificity for binding to miRNA precursors. The full molecular understanding of miRNA processing proposed herein will allow novel entry points for targeted manipulation of miRNA expression, which may have broad impacts on both basic and clinical science.